Single cell mutant selection for metabolic engineering of actinomycetes.

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting.

A computational toolset for rapid identification of SARS-CoV-2, other viruses and microorganisms from sequencing data.

In this paper, we present a toolset and related resources for rapid identification of viruses and microorganisms from short-read or long-read sequencing data. We present fastv as an ultra-fast tool to detect microbial sequences present in sequencing data, identify target microorganisms and visualize coverage of microbial genomes. This tool is based on the k-mer mapping and extension method.

Structural characterization of three noncanonical NTF2-like superfamily proteins: implications for polyketide biosynthesis.

Proteins belonging to the NTF2-like superfamily are present in the biosynthetic pathways of numerous polyketide natural products, such as anthracyclins and benzoisochromanequinones. Some have been found to be bona fide polyketide cyclases, but many of them have roles that are currently unknown. Here, the X-ray crystal structures of three NTF2-like proteins of unknown function are reported: those of ActVI-ORFA from Streptomyces coelicolor A3(2) and its homologs Caci_6494, a protein from an uncharacterized biosynthetic cluster in Catenulispora acidiphila, and Aln2 from Streptomyces sp.

Genetic Incorporation of Unnatural Amino Acids into Proteins of Interest in Streptomyces venezuelae ATCC 15439.

Site-specific, genetic incorporation of unnatural amino acids (UAAs) into proteins in living cells using engineered orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs is a powerful tool for studying and manipulating protein structure and function. To date, UAA incorporation systems have been developed for several bacterial and eukaryotic model hosts. Due to the importance of Streptomyces as prolific producers of bioactive natural products and as model hosts for natural product biosynthesis and bioengineering studies, we have developed systems for the incorporation of the UAAs p-iodo-L-phenylalanine (pIPhe) and p-azido-L-phenylalanine (pAzPhe) into green fluorescent protein (GFP) in Streptomyces venezuelae ATCC 15439.

Complete Genome Sequence of Streptomyces venezuelae ATCC 15439, Producer of the Methymycin/Pikromycin Family of Macrolide Antibiotics, Using PacBio Technology.

Here, we report the complete genome sequence of Streptomyces venezuelae ATCC 15439, a producer of the methymycin/pikromycin family of macrolide antibiotics and a model host for natural product studies, obtained exclusively using PacBio sequencing technology. The 9.03-Mbp genome harbors 8,775 genes and 11 polyketide and nonribosomal peptide natural product gene clusters.

Development of an Unnatural Amino Acid Incorporation System in the Actinobacterial Natural Product Producer Streptomyces venezuelae ATCC 15439.

Many Actinobacteria, most notably Streptomyces, produce structurally diverse bioactive natural products, including ribosomally synthesized peptides, by multistep enzymatic pathways. The use of site-specific genetic incorporation of unnatural amino acids to investigate and manipulate the functions of natural product biosynthetic enzymes, enzyme complexes, and ribosomally derived peptides in these organisms would have important implications for drug discovery and development efforts. Here, we have designed, constructed, and optimized unnatural amino acid systems capable of incorporating p-iodo-l-phenylalanine and p-azido-l-phenylalanine site-specifically into proteins in the model natural product producer Streptomyces venezuelae ATCC 15439.

Detection and Quantification of Ribosome Inhibition by Aminoglycoside Antibiotics in Living Bacteria Using an Orthogonal Ribosome-Controlled Fluorescent Reporter.

The ribosome is the quintessential antibacterial drug target, with many structurally and mechanistically distinct classes of antibacterial agents acting by inhibiting ribosome function. Detecting and quantifying ribosome inhibition by small molecules and investigating their binding modes and mechanisms of action are critical to antibacterial drug discovery and development efforts. To develop a ribosome inhibition assay that is operationally simple, yet provides direct information on the drug target and the mechanism of action, we have developed engineered E.

High-Quality Draft Genome Sequence of Actinobacterium Kibdelosporangium sp. MJ126-NF4, Producer of Type II Polyketide Azicemicins, Using Illumina and PacBio Technologies.

Here, we report the high-quality draft genome sequence of actinobacterium Kibdelosporangium sp. MJ126-NF4, producer of the type II polyketide azicemicins, obtained using Illumina and PacBio sequencing technologies. The 11.

Expanding our understanding of sequence-function relationships of type II polyketide biosynthetic gene clusters: bioinformatics-guided identification of Frankiamicin A from Frankia sp. EAN1pec.

A large and rapidly increasing number of unstudied "orphan" natural product biosynthetic gene clusters are being uncovered in sequenced microbial genomes. An important goal of modern natural products research is to be able to accurately predict natural product structures and biosynthetic pathways from these gene cluster sequences. This requires both development of bioinformatic methods for global analysis of these gene clusters and experimental characterization of select products produced by gene clusters with divergent sequence characteristics.

Emerging trends in the discovery of natural product antibacterials.

This article highlights current trends and advances in exploiting natural sources for the deployment of novel and potent anti-infective countermeasures. The key challenge is to therapeutically target bacterial pathogens that exhibit a variety of puzzling and evolutionarily complex resistance mechanisms. Special emphasis is given to the strengths, weaknesses, and opportunities in the natural product antibacterial drug discovery arena, and to emerging applications driven by advances in bioinformatics, chemical biology, and synthetic biology in concert with exploiting bacterial phenotypes.

Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids.

[Image: see text] Regions of the tyrosyl tRNA thought to interact with elongation factor Tu were randomized, and the resulting tRNA libraries were subjected to evolution. The tRNAs identified resulted in significantly improved unnatural amino acid-containing protein yields. In some cases, the degree of improvement varied in an amino acid-dependent manner.

One plasmid selection system for the rapid evolution of aminoacyl-tRNA synthetases.

We have developed a rapid, straightforward, one plasmid dual positive/negative selection system for the evolution of aminoacyl-tRNA synthetases with altered specificities in Escherichia coli. This system utilizes an amber stop codon containing chloramphenicol acetyltransferase/uracil phosphoribosyltransferase fusion gene. We demonstrate the utility of the system by identifying a variant of the Methanococcus jannaschii tyrosyl synthetase from a library of 10(9) variants that selectively incorporates para-iodophenylalanine in response to an amber stop codon.

Natural-product sugar biosynthesis and enzymatic glycodiversification.

Many biologically active small-molecule natural products produced by microorganisms derive their activities from sugar substituents. Changing the structures of these sugars can have a profound impact on the biological properties of the parent compounds. This realization has inspired attempts to derivatize the sugar moieties of these natural products through exploitation of the sugar biosynthetic machinery.

In vitro characterization of the enzymes involved in TDP-D-forosamine biosynthesis in the spinosyn pathway of Saccharopolyspora spinosa.

Forosamine (4-dimethylamino)-2,3,4,6-tetradeoxy-beta-D-threo-hexopyranose) is a highly deoxygenated sugar component of several important natural products, including the potent yet environmentally benign insecticide spinosyns. To study D-forosamine biosynthesis, the five genes (spnO, N, Q, R, and S) from the spinosyn gene cluster thought to be involved in the conversion of TDP-4-keto-6-deoxy-D-glucose to TDP-D-forosamine were cloned and heterologously expressed, and the corresponding proteins were purified and their activities examined in vitro. Previous work demonstrated that SpnQ functions as a pyridoxamine 5'-monophosphate (PMP)-dependent 3-dehydrase which, in the presence of the cellular reductase pairs ferredoxin/ferredoxin reductase or flavodoxin/flavodoxin reductase, catalyzes C-3 deoxygenation of TDP-4-keto-2,6-dideoxy-D-glucose.

Elucidation of the kijanimicin gene cluster: insights into the biosynthesis of spirotetronate antibiotics and nitrosugars.

The antibiotic kijanimicin produced by the actinomycete Actinomadura kijaniata has a broad spectrum of bioactivities as well as a number of interesting biosynthetic features. To understand the molecular basis for its formation and to develop a combinatorial biosynthetic system for this class of compounds, a 107.6 kb segment of the A.

Unusual sugar biosynthesis and natural product glycodiversification.

The enzymes involved in the biosynthesis of carbohydrates and the attachment of sugar units to biological acceptor molecules catalyse an array of chemical transformations and coupling reactions. In prokaryotes, both common sugar precursors and their enzymatically modified derivatives often become substituents of biologically active natural products through the action of glycosyltransferases. Recently, researchers have begun to harness the power of these biological catalysts to alter the sugar structures and glycosylation patterns of natural products both in vivo and in vitro.

Engineered biosynthesis of macrolide derivatives bearing the non-natural deoxysugars 4-epi-D-mycaminose and 3-n-monomethylamino-3-deoxy-D-fucose.

Previous pathway engineering work demonstrated that the desosamine biosynthetic pathway in could be converted to an efficient mycaminose biosynthesizing pathway by replacement of DesI with the hexose 3,4-ketoisomerase Tyl1a. In this work, FdtA, a ketoisomerase homologous to Tyl1a which catalyzes conversion of the Tyl1a substrate to the C-4 epimer of the Tyl1a product was used to replace DesI. The ability of desosamine pathway enzymes DesV, DesVI, DesVII, and DesVIII to accept substrates with inverted C-4 stereochemistry in the mutant expressing FdtA resulted in formation of macrolide derivatives bearing 4--D-mycaminose, a sugar heretofore unobserved in Nature.

Characterization of TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase from the D-mycaminose biosynthetic pathway of Streptomyces fradiae: in vitro activity and substrate specificity studies.

Deoxysugars are critical structural elements for the bioactivity of many natural products. Ongoing work on elucidating a variety of deoxysugar biosynthetic pathways has paved the way for manipulation of these pathways for the generation of structurally diverse glycosylated natural products. In the course of this work, the biosynthesis of d-mycaminose in the tylosin pathway of Streptomyces fradiae was investigated.

Glyco-stripping and glyco-swapping.

Glycosyltransferases (GTs) are ubiquitous enzymes that catalyze the transfer of a sugar moiety from an activated donor to an acceptor and thus play important roles in natural product biogenesis, virulence, and biomolecular recognition. Sugars are often critical for bioactivity of natural products, and methodologies for creating diverse glycoforms of these compounds are highly desirable. A recent study demonstrates that several GTs involved in natural product biosynthesis catalyze reversible reactions.

TDP-mycaminose biosynthetic pathway revised and conversion of desosamine pathway to mycaminose pathway with one gene.

Analysis of the tylosin gene cluster in Streptomyces fradiae uncovered an ORF, tyl1a, homologous to a hexose 3,4-isomerase found in Aneurinibacillus thermoaerophilus. Inclusion of the tyl1a gene along with other mycaminose biosynthetic genes (tylB, tylM1, tylM2, tylM3) identified in previous studies in an in vivo expression system successfully reconstituted the mycaminose pathway. Expression of tyl1a alone in the S venezuelae KdesI mutant converted a desosamine pathway to a mycaminose pathway.

Characterization of tylM3/tylM2 and mydC/mycB pairs required for efficient glycosyltransfer in macrolide antibiotic biosynthesis.

The heterologous expression of tylM3 and mydC, two homologous genes of previously unknown function, along with genes encoding their respective partner glycosyltransferases, tylM2 and mycB, and the necessary sugar biosynthesis genes significantly enhances the glycosyltransferase activity in the engineered Streptomyces venezuelae host in which the native glycosyltransferase, desVII, has been inactivated. Both glycosyltransferases accept the endogenous 12-membered macrolide, 10-deoxymethynolide, or the exogenously fed 16-membered macrolide, tylactone. Five new compounds were generated using this expression system.